Paul D. Shirk

European corn borer (Ostrinia nubilalis) induced responses enhance susceptibility in maize.

Herbivore-induced plant responses have been widely described following attack on leaves; however, less attention has been paid to analogous local processes that occur in stems. Early studies of maize (Zea mays) responses to stem boring by European corn borer (ECB, Ostrinia nubilalis ) larvae revealed the presence of inducible acidic diterpenoid phytoalexins, termed kauralexins, and increases in the benzoxazinoid 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one-glucose (HDMBOA-Glc) after 24 h of herbivory. Despite these rapidly activated defenses, larval growth was not altered in short-term feeding assays. Unexpectedly, ECB growth significantly improved in assays using stem tissue preconditioned by 48 h of larval tunneling. Correspondingly, measures of total soluble protein increased over 2.6-fold in these challenged tissues and were accompanied by elevated levels of sucrose and free linoleic acid. While microarray analyses revealed up-regulation of over 1100 transcripts, fewer individual protein increases were demonstrable. Consistent with induced endoreduplication, both wounding and ECB stem attack resulted in similar significant expansion of the nucleus, nucleolus and levels of extractable DNA from challenged tissues. While many of these responses are triggered by wounding alone, biochemical changes further enhanced in response to ECB may be due to larval secreted effectors. Unlike other Lepidoptera examined, ECB excrete exceedingly high levels of the auxin indole-3-acetic acid (IAA) in their frass which is likely to contact and contaminate the surrounding feeding tunnel. Stem exposure to a metabolically stable auxin, such as 2,4-dichlorophenoxyacetic acid (2,4-D), promoted significant protein accumulation above wounding alone. As a future testable hypothesis, we propose that ECB-associated IAA may function as a candidate herbivore effector promoting the increased nutritional content of maize stems.

Characterization of yolk proteins from the eggs of the indian meal moth, Plodia interpunctella

AMaet-The eggs of the Indian meal moth, Plodia inlerpuncrello (Hubner), contain four major yolk polypeptides (YPs). The four YPs were associated as two proteins that lacked immunocrossreactivity either as native proteins or as individual polypeptide subunits. Vitellin was found to be a glycolipoprotein composed of YPI (153 kDa) and YP3 (43 kDa) and had an apparent molecular mass ranging from 398 to 475 kDaas established by various methods. The other major yolk protein was composed ofglycosylated polypeptides YP2 and YP4 that were produced by ovarian tissues. Two forms of YP2/YP4 were observed under native conditions that had molecular masses of 93 and 235 kDa in pore-limited gel electrophoresis. The heteromneitv of the orotein mav be due to ~roteolvtic cleavaee of YP2 bv endorenous orateases. -. ~. smse polypept~dcs of 50 and 60 kDa appeared in solut~nnc conulnlng pan~all! pt.nlicJ YP2 YP4. dnd lhnc smaller polypcptidcr were shown to be related to YP? b) pcpt~de nl:applng. I hur. the )ulk 01 1. interpunaelh was found to contain two major yolk proteins; vitellin was identified as well as a large multiple subunit protein produced within the ovary that was unlike any previously described yolk protein from moths. Key Word Index: Indian meal moth, Plodio interpunctello, vitellin, egg proteins, yolk proteins

ALPHA -CRYSTALLIN PROTEIN COGNATES IN EGGS OF THE MOTH, PLODIA INTERPUNCTELLA: POSSIBLE CHAPERONES FOR THE FOLLICULAR EPITHELIUM YOLK PROTEIN

alpha-Crystallin protein cognates were found in germ cells of the Indianmeal moth, Plodia interpunctella (Shirk and Zimowska, 1997). A cDNA clone of 674 bp with a single open reading frame was isolated for a 25,000 molecular weight polypeptide member of this family, alpha CP25, and a single transcript of approximately 700 bp was found in the ovary of vitellogenic females. Both the DNA sequence and predicted amino acid sequence showed considerable homology with the embryonic lethal gene, l(2)efl, in Drosophila melanogaster. In addition to the sequence for l(2)efl, the predicted amino acid sequence for acp25 also showed significant sequence similarly with the alpha-crystallin A chain polypeptides from the lenses of vertebrae eyes. An N-terminal hydrophobic aggregation site and a C-terminal protective binding site common to alpha-crystallin proteins were present in the predicted acp25 and l(2)efl amino acid sequences, while only the C-terminal protective binding site was present in the small heat shock protein sequences from D. melanogaster. This evidence suggests that although the alpha-crystallin protein cognates in P. interpunctella evolved from a gene common with small heat shock protein genes, the amino acid sequence has converged on a structure similar to that of alpha-crystallin proteins. Native immunoblot analysis showed that the alpha-crystallin proteins formed high molecular weight complexes with the follicular epithelium yolk protein (FEYP) but not vitellin in yolk. An electroblot binding assay was used to show that the germ-cell alpha-crystallins of P. interpunctella bind specifically with the FEYP and that the binding was reversible in the presence of ATP or low pH. This evidence in conjunction with the evidence that the alpha-crystallins and FEYP form a stable complex that co-purifies from native egg proteins suggests that the alpha-cystallin cognates function as chaperones for the follicular epithelium yolk proteins in the embryos of P. interpunctella.

Comparison of Yolk Production in Seven Pyralid Moth Species

Summary The yolk proteins of six pyralid moths were analyzed and compared with the yolk proteins of Plodia interpunctella (Hubner). When cross-reacted in an Ouchterlony double immunodiffusion with antiserum raised to either total yolk proteins or purified vitellin from P. tnterpunctella, the yolk proteins of Anagasta kuehniella (Zeller), Cadra cautella (Walker), C. figulilella (Gregson), and Ephestia elutella (Hubner), closely related members of the subfamily Phycitinae, showed strong precipitation lines that consisted of four major yolk polypeptides (YPs). The yolk proteins from Amyelois transitella (Walker) were only weakly reactive, whereas yolk proteins from Galleria mel-lonella (L.) were not precipitated by either antiserum. Abdominal body walls (containing primarily fat body) from late pharate adult females were incubated in vitro and they secreted two major polypeptides that had molecular masses similar to the vitellogenins (YP1 and YP3) from P. interpunctella. In addition, ovarioles from late phar...

SYMPATRY OF ORIUS INSIDIOSUS AND O. PUMILIO (HEMIPTERA: ANTHOCORIDAE) IN NORTH CENTRAL FLORIDA

ABSTRACT Two closely related species of Anthocoridae, the minute pirate bugs Orius insidiosus (Say) and O. pumilio (Champion), were collected together from false Queen Annes lace/ large bullwort (Ammi majus) planted on an organic farm in Gainesville, Alachua Co., FL., over a period of 5 successive weeks. The presumptive prey on the false Queen Annes lace was a single species of thrips, Frankliniella bispinosa (Morgan). In the first 4 weekly collections from the flower heads, the densities of O. insidiosus adults were 2.7-fold higher than those of O. pumilio. No eggs or nymphs of either species were observed on the plants. Sex ratios (males:females) of 2.7 and 1.0 were observed for O. insidiosus and O. pumilio, respectively. A colony of O. insidiosus was established from field-collected specimens. The sex ratio of the F1 generation from this colony was 1.0, suggesting that the skewed field sex ratio was not a genetic phenomenon. These data demonstrate that these sympatric predators coexist at least temporarily, feeding on pests of the false Queen Annes lace.

Immuno-Fluorescent analysis of follicular growth and development in whole ovaries of the Indianmeal moth

The differentiation and growth of ovaries was analyzed using immuno‐fluorescence microscopy and then correlated with the changes in the external morphology of female pupae during metamorphosis of the Indianmeal moth, Plodia interpunctella (Hübner). Fourteen developmental points coincident with a daily change in the light:dark cycle were chosen for observation to describe the progress of cuticular, ovarian, and follicular development during metamorphosis. Follicular structure was examined in whole mounts of ovaries using an immuno‐fluorescent labelling technique. The growth of oocytes and nurse cell cap in terminal follicles was measured throughout ovarian development. A rapid increase in the relative size of the nurse cells began during the fourth scotophase and continued until the beginning of the sixth scotophase. Following the sixth scotophase, the relative size of the nurse cells decreased until they disintegrated prior to choriogenesis. Oocytes began to grow rapidly during the fifth scotophase, coincident with the initiation of vitellogenesis, and continued to grow until choriogenesis was initiated just after adult eclosion. The rate of follicular growth was related to the position of the follicle in the ovariole; the closer to the terminal position, the greater the rate of growth. Thus, at adult eclosion, each ovariole contained a linear array of follicles in progressive stages of development with the terminal follicles ready to begin choriogenesis.

Predator-Prey Relationships on Apiaceae at an Organic Farm

ABSTRACT Orius insidiosus (Say) and O. pumilio (Champion) were confirmed to be sympatric in north central Florida as the major predators of the Florida flower thrips, Frankliniella bispinosa (Morgan), on flowers of Queen Annes lace, Daucus carota L. and false Queen Annes lace, Ammi majus L. F. bispinosa was the predominant thrips observed on both flowers but colonized D. carota to a greater extent and earlier in the season than A. majus. Despite differences in the abundance of F. bispinosa on the two plants, neither Orius species showed host plant affinities. Population profiles for the thrips and Orius spp. followed a density dependent response of prey to predator with a large initial prey population followed by a rapid decline as the predator populations increased. The temporal increases in Orius spp. populations during the flowering season suggest that they were based on reproductive activity. As observed in a previous study, O. insidiosus had a larger population than O. pumilio and also had a predominantly male population on the flowers. By examining carcasses of the prey, there appeared to be no sexual preference of the thrips as prey by the Orius spp. as the prey pattern followed the demographics of the thrips sex ratio. Few immatures of either thrips or Orius spp. were observed on D. carota or A. majus, which suggests that oviposition and nymphal development occurred elsewhere. Based on these findings, D. carota and A. majus could serve as a banker plant system for Orius spp.

Identity of Two Sympatric Species of Orius (Hemiptera: Heteroptera: Anthocoridae)

Abstract The minute pirate bugs, Orius insidiosus (Say) and Orius pumilio (Champion) (Hemiptera: Heteroptera: Anthocoridae), are closely related species known to be sympatric in north Florida. Here, male and female genitalia, DNA sequences, and the effects of within- and between-species pairings on egg production and egg development were examined to develop a better understanding of the relationship between these two species. Interspecific matings between the two species did not result in viable progeny. Although there were gross similarities in the morphology of the male parameres (external genitalia) between the two species, the cone in O. pumilio was much broader with a greater spiral twist and the flagellum was longer than in O. insidiosus. Correspondingly, there were differences in the morphology of the copulatory tubes of the females of the two species. In O. insidiosus, the organ was somewhat longer than in O. pumilio and oriented parallel to the abdominal midline, while the copulatory tube in O. pumilio tilted slightly towards the midline. Additionally, the copulatory tube for O. pumilio included a sclerotized basal mound that was not present in O. insidiosus. These morphological differences suggest that successful copulation between these species could be difficult. In contrast to conspecific matings, interspecific matings resulted in few or no eggs laid over a period of two weeks and no viable progeny. Comparison of the 18S ribosomal gene ITS-1 sequences between the two species demonstrated only 91% homology. When yolk protein contents were examined to determine whether reproductive physiology had shifted to full egg production, interspecifically mated females contained amounts of yolk protein comparable to that in fed, but unmated females; this was less than 10% of the yolk protein previously found in fed and conspecifically mated females. These findings together confirm that O. insidiosus and O. pumilio are indeed two separate species.

Declining ecdysteroid levels are temporally correlated with the initiation of vitellogenesis during pharate adult development in the Indianmeal moth, Plodia interpunctella

Photo-synchronized pupae were used to determine the titer of ecdysteroids during the pupal and pharate adult stages of the Indianmeal moth, Plodia interpunctella (Hiibner). For female pupae that were kept in a 16h 1ight:S h dark (longday) photocycle, there was one major peak of ecdysteroids. The ecdysteroids reached a maximum of 2000-2500 pg/mg wet wt between 28-36 h after pupation. The ecdysteroids declined to 400 pglmg wet wt by 68 h after pupation. For pupae that were kept in continuous darkwss, the ecdysteroid peak became broader with a maximum at 24 h (3130 pg/mg wet wt). The composition of ecdysteroids at various times during pharate adult development was determined usiug gas chromatographass spectrometry with selected ion monitoring (GC-MS (S1M)I. Eedysone was the major component (93%) in samples taken 28 h after pupation, at the maximum of the pupal peak. Thus, high levels of ecdysoue are correlated with the beginning of adult ovarian development. As pharate development progressed, the proportion of 20-hydroxyecdysow increased so that by 100 h after pupation 20-hydroxyecdysone was the only ecdysteroid detected. Previously, the initiation of vitellogenesis was determined to begin between % and 100 h after pupation. In conjuction with previous work, these findings demonstrate that the ecdysteroid titers decline before viteUogenesis is initiated and egg maturation can be completed. Ecdysteroids Vitellogenesis Metamorphosis Radioimrnunoassay GC-MS quantification

5′ coding region of the follicular epithelium yolk polypeptide 2 cDNA in the moth, Plodia interpunctella, contains an extended coding region†

The 5 region of YP2 cDNA, a follicular epithelium yolk protein subunit in the moth, Plodia interpunctella, shows that the polypeptide contains an extended internal coding region. Partial cDNA clones for YP2 were isolated from a pharate adult female ovarian cDNA expression library in Lambda Zap II by screening with antigen selected YP2 antiserum. The 5 sequence of the YP2 transcript was determined by 5 RACE PCR of ovarian mRNA using YP2 sequence-specific nested primers. The combined cDNA and 5 RACE sequencing showed the YP2 transcript to be 1971 bp in length up to the poly(A) tail with a single open reading frame for a predicted polypeptide of 616 amino acids. Northern analysis showed a single YP2 transcript to be present in ovarian RNA that was approximately 2 kb in length. The predicted amino acid sequence for YP2 from P. interpunctella is most closely related to egg specific protein (ESP) from Bombyx mori and the partial YP2 sequence from Galleria mellonella. YP2 from P. interpunctella also is similar to vertebrate lipases and contains a conserved lipid binding region. However, the 5 coding region of YP2 from P. interpunctella contains an in-frame insert of approximately 438 bp that had replaced an approximately 270-bp region as compared with ESP from B. mori and YP2 of G. mellonella. This suggests that the insert occurred by a recombinational event internal to the YP2 structural gene of P. interpunctella.